JP4242647B2 - Enzymatic deprotection of amines and hydroxides - Google Patents

Description

    The present invention relates to a mild method of using an enzyme that removes amine and hydroxide protecting groups.

During organic synthesis, N-carbobenzyloxy (N-CBZ) groups are generally used to protect amino and hydroxide groups. Other similar “carbamate” protecting groups are also used to protect amino groups. Chemical deprotection is usually performed by a method such as hydrogenation using a palladium catalyst. However, if there are other groups that are susceptible to deprotection conditions (eg sulfur during hydrogenation), another deprotection method is required.
(Summary of Invention)

  It has been found that microorganisms can be easily isolated from soil samples using the selection technique of the present invention to provide effective enzymatic activity to specifically release such protecting groups. That is, an enzymatic method of deprotection performed under mild conditions (eg, aqueous medium at room temperature and atmospheric pressure) can be used, which avoids harm to either sensitive or potentially sensitive groups.

The present invention has the formula:
ArC ^* (R) H— (CH ₂ ) n —O—C (═O) —
[Wherein each substituent has the same meaning as described below]
A method for deprotecting a hydroxide or amine protected with a group comprising contacting the protected hydroxide or amine with an enzyme effective for elimination of the protecting group; and then recovering the amine Provide deprotection law.

  The present invention also relates to a method for isolating a bacterium producing an enzyme effective for elimination of a protecting group, which comprises a medium having a growth selective amount of amine compound protected as described above. Also provided is an isolation method characterized by growing the expected bacteria; then isolating the bacteria that grow on the medium.

［式中、Ｐr−は上記の保護基である］ The present invention further provides a method for resolving the desired enantiomer of an amine or hydroxide attached to a chiral carbon. Amines or hydroxides protected with such groups are stereo-specifically hydrolyzed by the method of the present invention. The desired enantiomer is either hydrolyzed or resistant to hydrolysis.
In one embodiment, the contacting step realizes the following reaction:

[In the formula, Pr- is the above-mentioned protecting group]

In other embodiments, the contact achieves the following reaction:

In yet another embodiment, the contact achieves the following reaction:

(Details of the invention)
The present invention has the formula:
It can be used for elimination of a number of carbamate protecting groups, represented by ArC ^* (R) H— (CH ₂ ) n —O—C (═O) —. Here, R is H or independently the same as Ar, and n is 0 or 1-4. Ar refers to an aromatic or heteroaromatic ring having 5 to 6 ring atoms and 1 to 2 heteroatoms selected from O, N or S. Ar is independently Ar, except that it is not substituted with amino, alkanoyloxy, alkoxy, alkyl, alkylamino, allyl, carboxy, cycloalkyl, halo, haloalkyl, hydroxy, hydroxyalkyl or nitro, or (a) further aryl. May be substituted with up to one group of Ar ^* , (b) Ar ^* -alkyl- or (c) Ar ^* O- which is the same as

The ring atom of Ar adjacent to C ^* is a group of —CH ₂ —, —O—, —NH—, —S (O when R is Ar, q is 0 or 1-2 and r is 0 or 1-2. ) q- or -P (O) r- can be substituted to form a bond bridge at the corresponding position on R. In one embodiment, when R is H, n is 0. In other embodiments, n is 1 when R is the same as Ar. As shown in the examples below (see Table 2), the method is stereospecific and can be used to resolve racemic mixtures.

  These protecting groups are 9-fluorenylmethyl carbamate, 9- (2-sulfo) fluorenylmethyl carbamate, 9- (2,7-dibromo) fluorenylmethyl carbamate, 2,7-di-t-. Butyl- [9- (10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl) methyl carbamate, benzyl carbamate, p-methoxybenzyl carbamate, p-nitrobenzyl carbamate, p-bromobenzyl carbamate, p -Chlorobenzylcarbamate, 2,4-dichlorobenzylcarbamate, 9-anthrylmethylcarbamate, diphenylmethylcarbamate, m-chloro-p-acyloxybenzylcarbamate, p- (dihydroxyboryl) benzylcarbamate, 5-benzisoxazolyl Methylcarbame 2- (trifluoromethyl) -6-chromonylmethyl carbamate, m-nitrobenzyl carbamate, 3,5-dimethoxybenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, S-benzylthiocarbamate, Illustrated by compounds such as p-cyanobenzylcarbamate, 2-furanylmethylcarbamate, 4- (trimethylammonium) benzylcarbamate and 2,4,6-trimethylbenzylcarbamate.

Protecting groups such as these are described in standard texts such as “Protective Groups in Organic Synthesis” by Greene and Wuts (Shon Willy and Sons, New York, 1991) (especially pages 315-348).
The alkyl component of the substituent is C ₁ -C ₆ or C ₂ -C ₆ where the C ₁ component is chemically unsuitable (eg in the case of alkanoyl). Cycloalkyl groups _are C 3 -C _6. Haloalkyl preferably refers to perhaloalkyl, preferably trifluoromethyl. Halo is preferably chloro or fluoro.

  In one embodiment, the carbamate protecting group is a phenylmethyloxycarbonyl group, where phenyl is substitutable. Substitutions to phenylmethyloxycarbonyl are, for example, those listed for Ar above.

  The source of the enzyme used in the present invention can be isolated as an isolated bacterium having an appropriate activity. The method of isolation is preferably sorting by growth in culture medium, wherein only nitrogen that supports sufficient growth from the amine compound in which the amine is protected with the carbamate protecting group in question, or a related carbamate protecting group. Can only get. The examples below demonstrate that such bacteria can be isolated from very common sources of bacteria, such as environmental or soil samples.

  The examples below illustrate that the inventor's identified sorting technique is effective in isolating appropriate bacteria in routine experimentation, thereby isolating the appropriate enzyme source. The examples relate to bacteria isolated by selection of CBZ-protected nitrogen source growth. Thus, this example establishes the inventor's understanding, that is, the same approach using a protecting group that is compatible with the protecting group that is desired to be eliminated, and that the appropriate enzyme can be collected without undue experimentation. To do.

  When an amine or hydroxide that entails enzymatic desorption is identified as the most promising candidate for a cause that is resistant to cleavage by a given enzyme of the proposed substrate, the amine Appropriately protected versions of (or analogs, or amine analogs of hydroxides) can be used to screen for different bacteria, and therefore for different enzymes. Where surrogate enzymes are required, collections of bacterial cultures that produce different deprotection enzymes or each useful enzyme can be stored and screened.

  If the amine or hydroxide to be protected and deprotected is a complex molecule with an amine or hydroxide moiety that binds to a relatively remote component, the amine model used in the screening method is adjacent to the amine or hydroxide. Can be modeled into a complex part. It is preferred to take care that the components derivatized in the complex molecule are similarly derivatized nearby.

  As shown below, deprotection provided by the present invention can be carried out using whole bacterial cells, whole cell extracts, or purified enzyme preparations. Enzymes can be used catalytically because they can generally be used in small quantities and impurities provided by the enzyme source (eg, less pure) should not produce significant amounts of material that should behave like the intended product. Works.

  That is, the impurities imparted by the enzyme source are quickly selected in preparation for workup after the reaction. In particular, when using extracts, the impurities are generally polymeric and the typical intended product is generally not polymeric so that the impurities are quickly sequestered from the product.

  Also, as shown below, the substrate used in the enzyme selection method is a convenient means of measuring enzyme activity and thus isolating the enzyme by selective microbiological enrichment and traditional protein chemistry techniques. Is granted.

  The amine or hydroxide that is protected with a protecting group may be any amine or hydroxide on any molecule. In many embodiments, the amine or hydroxide on the molecule is sized according to non-repetitive synthesis techniques (of course, the deprotection techniques of the present invention are repetitive techniques such as those used in peptide or nucleic acid synthesis). Can also be used).

In one preferred embodiment, the amine or hydroxide is part of a bioactive agent that is bioavailable to an animal after ingestion, or part of the precursor of such a life agent. .
In one aspect, the amine is preferably an α- or β-amino acid, most preferably an α-amino acid.

  Examples of amines include alanine, valine, leucine, isoleucine, proline, 4-hydroxyproline, phenylalanine, tryptophan, methionine, glycine, serine, homoserine, threonine, cysteine, homocysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine Α-amino-ε-caprolactam (lysine lactam), α-amino-δ, δ-dimethyl-ε-caprolactam, ε-methyllysine, ornithine, arginine, histidine or 3-methylhistidine, or an amine thereof, The alkyl moiety is substituted with hydroxy or alkyl, the amino is substituted with up to one alkyl, or the phenyl moiety is substituted with the groups listed for Ar above It may be.

  Such amino acids may be L or D amino acids. In addition, such amino acids are derivatized to form part of a larger molecule through a bond formed by a dehydration reaction with an amine or carboxylic acid component, or by a carbon-nitrogen bond formed in the amine component. can do.

［式中、ｍは０または１；ＹはＣＨ_２、Ｓ−(Ｏ)tまたはＯ、但し、ｍが１のときのみ、ＹはＳ−(Ｏ)tまたはＯ；ＸはＳ−(Ｏ)tまたはＯ；ｎは１または２；ｔは０、１または２；Ｒ_３は水素、アルキル、置換アルキル、アリール−(ＣＨ_２)p−；およびｐは０または１〜６である］ Another type of alpha amino acid particularly useful in the present invention is shown by the following formula:

[Wherein, m is 0 or 1; Y is CH ₂ , S— (O) t or O, provided that only when m is 1, Y is S— (O) t or O; X is S— (O ) t or O; n is 1 or 2; t is 0, 1 or 2; R ₃ is hydrogen, alkyl, substituted alkyl, aryl- (CH ₂ ) p—; and p is 0 or 1-6]

Among these amines, those of the following formula are particularly preferred amines.

  These compounds are described in US Pat. 5508272 is described in more detail. The teachings regarding the preparation and use of these compounds in the patent are incorporated by reference. Additional compounds of particular interest for the use of the present invention include WO 00/47207 and US Patent No. No. 555297. The teachings relating to the production and use of the compounds described in these patent documents are incorporated by reference.

Protected amines or hydroxides are generally
ArC ^* (R) H— (CH ₂ ) n —O—C (═O) —X
[Wherein X is a leaving group, bromo, chloro, tosyl]
Is reacted with the corresponding amine or hydroxide. ArC ^* (R) H— (CH ₂ ) n —O—C (═O) —X is, for example, ArC ^* (R) H— (CH ₂ ) n —OH converted to phosgene, carbomil diidazole, triphosgene or comparable reagents. It is formed by reacting with.

Definitions Each of the terms shown below has the following significance for the purposes of the present invention.
Bioactive agent:
A bioactive agent is a substance such as a chemical substance that can act on a cell, virus, tissue, organ or organism, such as but not limited to a change in the function of a cell, virus, organ or organism. Pesticides or drugs (ie pharmaceuticals) that cause The organism is preferably a mammal, more preferably a human.

Medium with growth selective amount of amine compound:
A medium having a growth-selective amount of a protected amine compound is a medium in which the amount of other amines other than the amine compound is less than an amount effective to promote bacterial growth in a growth-mediated selection method. Preferred protected amines are essentially a single nitrogen source.

(Example)
Example 1: Selection Technique for Microorganism Isolation A selective culture technique is used to isolate microorganisms that can utilize N-α-CBZ-L-lysine as the sole source of nitrogen. Collect soil samples from various sites in New Jersey. About 1 g of soil sample is suspended in 5 mL of water and mixed well to allow the sample to settle. The upper layer solution of various samples was added to A medium containing 1% N-α-CBZ-L-lysine (glucose 2%, KH ₂ PO ₄ 0.2%, K ₂ HPO ₄ 0.2%, MgSO ₄ 0.01. %, FeSO ₄ 0.001%, ZnSO ₄ 0.001%, pH 7.0).

After 4 days of growth, if the medium becomes cloudy, transfer the culture to the above medium containing 1.5% agar in a Petri plate. From this enrichment culture technique, eight colonies are isolated. One culture (Z-2) was further identified as Sphingomonas paucimobilis strain, which was designated as Sphingomonas paucimobilis strain ATCC202027 (American Type Culture Collection, Rockville, Maryland)
Deposited with American Type Culture Collection. This culture (fungus) is used as a source of CBZ-deprotecting enzyme.

Example 2 Growth of Sphingomonas paucimobilis N-α-CBZ-L-phenylalanine or [4S- (4α, 7α, 10aβ)]-octahydro-5-oxo-4-[[( Phenylmethoxy) carbonyl] amino] -7H-pyrido- [2,1-b] [1,3] thiazepine-7-carboxylic acid methyl ester (Compound A). The Sphingomonas paucimobilis culture is inoculated into A medium containing 1% N-α-CBZ-L-phenylalanine or 1% Compound A. After 2 days of growth, transfer the cultures to A medium containing 1% N-α-CBZ-L-phenylalanine or 1% BMS199541 and 1.5% agar in petri plates.

Colonies isolated from Petri plates were isolated from B medium containing 1% N-α-CBZ-L-phenylalanine and / or 1% Compound A (yeast extract 0.015%, glucose 2%, KH ₂ PO ₄ 0.2). %, K ₂ HPO ₄ 0.2%, MgSO ₄ 0.01% and NaCl 0.1%, pH 7). The culture is grown on a rotary shaker at 28 ° C. and 280 RPM for 24 hours. This culture is placed in a vial (1 mL of culture in a 2 mL vial) and stored at -70 ° C for future use.

  One vial (containing 1 mL of Sphingomonas paucimobilis in B medium) is used to inoculate 100 mL of B medium. The culture is grown on a rotary shaker at 28 ° C. and 280 RPM for 48 hours. Cells are harvested by centrifugation at 18000 × g for 15 minutes and stored at −70 ° C. for future use.

Example 3: Biotransformation using whole cells In this method, Sphingomonas paucimobilis is grown in 25 mL B medium containing 25 mg substrate (Compound A or CBZ-L-phenylalanine) in a 250 mL flask. The flask is incubated on a shaker at 28 ° C. and 250 rpm. After 48 hours of biotransformation, the cells are removed by centrifugation.

The product [4S- (4α, 7α, 10aβ)]-octahydro-5-oxo-4-amino-7H-pyrido- [2,1-b] [1,3] thiazepine-7-carboxylic acid methyl ester ( The upper layer solution containing compound B) or L-phenylalanine is analyzed by HPLC. The results are shown in Table 1 below.
Table 1:
Substrate product conversion (%)
Compound A Compound B 100
CBZ-L-L-phenylalanine 100
Phenylalanine

HPLC analysis:
HPLC analysis is performed using a Hewlett-Packard (HP) 1090 instrument with a Vydac C-18 reverse phase column. Mobile phase, solvent A containing 0.1% trifluoroacetic acid (TFA) in water and solvent B containing 0.1% TFA in 70% acetonitrile / 30% water. The following solvent A and B gradients are used to separate the substrate and product.

  0 min: 100% A, 0-15 min: 50% B, 15-25 min: 100% B, 25-26 min: 0% B, and 26-30 min: 0% B. The flow rate is 1 mL / min. The column temperature is ambient and the detection wavelength is 215 nm. Under these conditions, the retention times of Compound A, Compound B, CBZ-L-phenylalanine and L-phenylalanine are 15.48 minutes, 7.28 minutes, 16.99 minutes and 7.35 minutes, respectively. All other CBZ-containing compounds were also analyzed using these conditions.

Example 4: Sphingomonas paucimobilis
Deprotection of CBZ using cell extract of ATCC 202027 Preparation of cell extract of Sphingomonas paucimobilis ATCC 202027:
Cell extract preparation is performed at 4-7 ° C. Cells were washed with 50 mM potassium phosphate buffer (pH 7.0) and washed cells (100 g) containing 500 mL Buffer A (50 mM phosphate buffer, pH 7.0, 10% glycerol and 2 mM DTT). ).

  To this cell suspension is added 1 mM phenylmethylsulfonyl fluoride (PMSF) in isopropanol. The cell suspension (20% W / V, wet cells) is passed through a microfluidizer (Microfluidizer, Microfluidics, Inc.) at 12000 psi (2 passes) and the dissociated cells are 4 × 25000 × g. Centrifuge for 30 minutes at ° C. The upper layer liquid obtained after centrifugation is referred to as a cell extract.

CBZ-deprotection using cell extract:
Cell extracts are used for deprotection of CBZ-groups from various compounds. It is useful for deprotecting the CBZ-group in various ways. Various D and L-CBZ-protected amino acids are cultured with cell extracts at 42 ° C. for 18-20 hours. The reaction is stopped by adding 2 volumes of 50% acetonitrile containing 0.4% trifluoroacetic acid (TFA). From the results shown in Table 2 below, it can be seen that the enzyme is specific for hydrolyzing the CBZ-group of the CBZ-protected L-amino acid.

Table 2:
Substrate product conversion (%)
N-α-CBZ-L-tyrosine L-tyrosine 100
N-α-CBZ-D-tyrosine D-tyrosine 1.58
O-α-CBZ-L-tyrosine L-tyrosine 100
N-α-CBZ-L-leucine L-leucine 100
N-α-CBZ-D-leucine D-leucine 1.2
N-α-CBZ-L-phenylalanine L-phenylalanine 100
N-α-CBZ-D-phenylalanine D-phenylalanine 0
N-α-CBZ-L-lysine L-lysine 52
N-ε-CBZ-D-lysine D-lysine 7
N-α-ε- (CBZ) ₂ -L-lysine L-lysine 24
N-α-CBZ-L-proline L-proline 100
N-α-CBZ-D-proline D-proline 0
Compound A Compound B 95

Example 5: Purification of CBZ-deprotection enzyme and use of the purified enzyme in deprotection of CBZ-group from CBZ-containing compounds
Enzyme assay 0.5 mg of compound A or CBZ-phenylalanine is cultured in 50 mM phosphate buffer (pH 7) at 45 ° C. with 0.4 mL of cell extract / fraction for 18 hours. The reaction is stopped by adding 1 mL of 50% acetonitrile containing 0.4% TFA. Filter sample and analyze product and starting material by HPLC

Protein Assay Protein concentration is measured using the Bio-Rad protein assay. The assay was performed according to the manufacturing (Bio-Rad) protocol.
Enzyme purification All purification steps are performed at room temperature. The enzyme is purified using CBZ-L-phenylalanine as a substrate.

  The cell extract prepared above is adsorbed batchwise for 2 hours with DEAE-cellulose (previously equilibrated with buffer A). The follow-through containing the active enzyme is precipitated with ammonium sulfate (516 g / L) for 2 hours with constant stirring. The resulting precipitate obtained by centrifugation (15000 rpm, 4 ° C.) is solubilized in buffer A containing 1M ammonium sulfate and phenylsepharose (20 mL column pre-equilibrated with buffer A containing 1M ammonium sulfate). To load.

  The column is washed successively with buffer A containing 1M ammonium sulfate, 0.5M ammonium sulfate and 0.2M ammonium sulfate. Finally, the enzyme is eluted with buffer A. Fractions containing active enzyme are pooled (30 mL) and concentrated on an Amicon PM-10 membrane (8 mL). The enzyme is then loaded onto an S-200 gel-filtration column (400 mL column).

  The enzyme is eluted with buffer A at a flow rate of 0.8 mL / min. In these steps, the enzyme is purified 150 times or more, and the specific activity in this case is 13.9 units / mg protein (Table 3 below). Units are defined as μmol formed product / min / mg protein. The enzyme is a dimeric protein having a molecular weight of -154,000 daltons and a subunit molecular weight of 45,000 daltons as determined by SDS-PAGE.

Table 3: Purification of CBZ-deprotecting enzymes
Volume Activity Protein Specific activity Purification
Step mL U / mL mg / mL U / mg multiple Cell extract 500 0.142 1.8 0.08 1.00
DE52-Follow-through 700 0.182 0.58 0.32 4.00
Ammonium sulfate precipitation 60 2.496 7.45 0.34 4.25
Phenyl Sepharose column 28 0.117 0.13 0.90 11.41
S-200 gel-filtration column 7 0.139 0.01 13.90 176.20

The purified enzyme prepared according to the description in this section was used to deprotect the CBZ-containing compounds shown in Table 4 below.
Table 4:
Substrate product conversion rate (%)
Compound A Compound B 100
CBZ-L-phenylalanine L-phenylalanine 100

Example 6: Enzymatic deprotection of Compound A in a 250 mg prep batch Cell extracts are prepared as described in the section above. To 250 mL of cell extract, 250 mg of Compound A is added and cultured at 28 ° C. and 95 rpm. After 40 hours of reaction, 250 mL of acetonitrile is added. Substrate and product are analyzed by HPLC. The molar yield of compound B was 87%.

Example 7: Enzymatic deprotection of CBZ-containing compounds A cell extract prepared from Sphingomonas paucimobilis ATCC 202027 was used as described in the previous section and [(3S) -hexahydro-2-oxo-1- [2-oxo- 2- (1-Pyrrolidinyl) ethyl] -1H-azepin-3-yl] carbamic acid phenylmethyl ester (Compound C) was deprotected to give (S) -1-[(3-aminohexahydro-2-oxo -1H-azepin-1-yl) acetyl] pyrrolidine (Compound D) is formed.

Example 8: Enzymatic deprotection of CBZ-containing compounds 6-[(Phenylmethoxy) carbonyl] amino] hexahydro-2,2-dimethyl using a cell extract prepared from Sphingomonas paucimobilis ATCC 202027 as described in the previous section. -7-oxo-1H-azepine-1-acetic acid ethyl ester / hydrochloride 1 is deprotected to give 6-aminohexahydro-2,2-dimethyl-7-oxo-1H-azepine-1-acetic acid ethyl ester It is referred to as hydrochloride (compound E).

  As a note, for publications and references including, but not limited to, patents and patent applications cited herein, these individual publications and references are hereby fully described. The entire cited portion is hereby incorporated by reference in its entirety, even if it is clearly and individually indicated as being introduced in the document. Any patent application that serves as a basis for priority claim of this application is incorporated herein by reference, as well as the above-mentioned publications and references.

  While this invention has been described with emphasis on preferred embodiments, it should be understood that variations in preferred inventions and methods may be used and that the invention may be practiced otherwise than as specifically described herein. The intention is obvious to those skilled in the art. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as defined by the appended claims.

Claims (9)

formula:
ArC ^* (R) H— (CH ₂ ) n —O—C (═O) —
[Wherein R is H or independently the same as Ar, and n is 0 or 1 to 4, Ar represents an aromatic or heteroaromatic ring having 5 to 6 ring atoms, The heteroaromatic ring has 1 to 2 heteroatoms selected from O, N or S and is amino, alkanoyloxy, alkoxy, alkyl, alkylamino, allyl, carboxy, cycloalkyl, halo, haloalkyl, hydroxy, hydroxy Up to one group of Ar ^* , (ii) Ar ^* -alkyl- or (iii) Ar ^* O-, independently of alkyl or nitro, or (i) independently substituted with further aryl The ring atom of Ar adjacent to the ring atom bonded to C ^* is —CH when R is Ar, q is 0 or 1-2 and r is 0 or 1-2. _2— , —O—, —NH—, —S (O) q— or —P (O) r— can be substituted to form a bond bridge at the corresponding position on R]
A method for deprotecting a hydroxide or amine protected with a group of
A deprotection process characterized in that a protected hydroxide or amine is contacted with an enzyme obtained from Sphingomonas pausobibilis to directly remove the protecting group; and then the hydroxide and amine are recovered.   The deprotection method according to claim 1, wherein the protecting group is a phenylmethyloxycarbonyl group which can be substituted.   The deprotection method according to claim 1, wherein when R is H, n is 0.   The deprotection method according to claim 1, wherein n is 1 when R is the same as Ar.   The deprotection method according to claim 1, wherein the enzyme is obtained from Sphingomonas pausobibilis strain ATCC2020027. The protective compound is alanine, valine, leucine, isoleucine, proline, 4-hydroxyproline, phenylalanine, tryptophan, methionine, glycine, serine, homoserine, threonine, cysteine, homocysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine , [Alpha] -amino- [epsilon] -caprolactam (lysine lactam), [epsilon] -methyllysine, ornithine, arginine, histidine or 3-methylhistidine, or these amines, the alkyl part of which is substituted with hydroxy or alkyl The amino is substituted with up to one alkyl, the phenyl component is alkyl, alkanoyloxy, alkoxy, amino, carboxy, cycloalkyl, halo, hydride Alkoxy, via a bond formed by the dehydration reaction with either amines or carboxylic acid component Ar ^* or Ar * ^O-in but substituted, or carbon is formed on the amine component - by nitrogen bond, more The deprotection method according to claim 1, which is a derivative of the amine that forms part of a larger molecule.   The deprotection method according to claim 6, wherein the amine is α-amino-ε-caprolactam or α-amino-δ, δ-dimethyl-ε-caprolactam, or a derivative thereof. The deprotection method according to claim 1, wherein the contact between the protected hydroxide or amine and the enzyme causes the following reaction.

Wherein, Pr- is ^{ArC * (R) H- (CH} 2) n-O-C (= O) - is] The reaction

[Wherein CBZ- is N-carbobenzyloxy]
The deprotection method according to claim 8.